I want to be isolate nitrogen fixing green sulfur bacteria.which technique apply i quickly identify green sulfer bacteria.
16 s r RNA sequencing with group specific primers. Isolation and purification of green sulfur bacteria is very difficult. Through group specific primers, you can identify GSB in mixed cultures also.
Cultures should be grown routinely in medium Pf-7, a modification of Pfennig's
sulfur phototroph medium , which contains both sulfide (2.5 mM) and thiosulfate (10 mM) as photosynthetic electron donors and acetate plus CO2 as carbon sources. For growth on N2, medium Pf-7 is modified as follows: sodium acetate (6.5 mM) was substituted for ammonium acetate, NH4C1 was omitted, sulfide is increased to 5 mM, and 10 mM 3-N-morpholine propanesulfonic acid (MOPS) is added per liter (pH 7). This modified medium Pf-7 is referred to as medium Pf/N2.
Growth conditions. Cultures are grown photosynthetically on ammonia or glutamine as nitrogen sources in screwcapped tubes or bottles. For growth on N2, cultures are grown in 250-ml Erlenmeyer sidearm flasks containing 120 ml of medium Pf/N2, a small magnetic stirring bar, and a headspace of N2-H2 (99:1). Cultures are routinely inoculated (3 to 4% volume) in an anaerobic chamber N2-grown cells are agitated very gently (but not actively stirred) by action of the small stir bar drive.
Once you grow the bacteria then you have to do 16 S rRNA sequencing analysis and DNA-DNA hybridisation to compare to the most related species.
Some descriptions related to the enrichment, isolation and characterization of Green Sulfur Bacteria could be found for instance in the following publication (where I participated):
- Novel green sulfur bacteria phylotypes detected in saline environments: ecophysiological characters versus phylogenetic taxonomy (DOI 10.1007/s10482-010-9420-x)
For identification purposes please refer to the Phylogenetic taxonomy previously described by Imhoff:
Phylogenetic taxonomy of the family Chlorobiaceae on the basis of 16S rRNA and fmo (Fenna– Matthews–Olson protein) gene sequences (DOI 10.1099/ijs.0.02403-0)
Also consider the following manuscript when aiming to describe a new isolate:
Recommended standards for the description of new species of anoxygenic phototrophic bacteria (DOI 10.1099/ijs.0.03002-0)
Best wishes
Dear Islam,
PFENNIG'S medium is found to be suitable in this purpose. You may have a look in the attached document for details.
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